Genome-wide Identification Of MAPKKK Gene Family In Arabidopsis Pumila And Transgenic Study Of ApMEKK18-2

Ephemeral plants are widely distributed in the desert areas of Xinjiang and play an important role in wind and sand control,soil and water conservation,and improvement of micro-ecological environment.They are well-known as pioneer plants of community succession.Arabidopsis pumila,an Ephemeral plant of the Brassicaceae family,grows in the arid and saline desert areas of northern Xinjiang.It exhibits excellent tolerance to various abiotic stresses,such as salt damage,cold damage,and drought,and it contains a wealth of resistance genes.China is the world’s third-largest country with saline-alkali land,with a total area of approximately 9.9×10~7 hm~2.Improving the utilization rate of saline-alkali land plays an important role in the development of China’s agriculture.Therefore,exploring and utilizing the genetic resources of A.pumila and breeding salt-resistant crops through genetic engineering are of great significance for the improvement and utilization of saline lands.Mitogen-activated protein kinase kinases(MAPKKKs)are important components of the MAPK cascade and play key roles in developmental processes and stress responses.Genome-wide identification of MAPKKK gene family members in A.pumila and genetic engineering of ApMEKK18-2 gene were carried out in this study.The main research results are as follows:1.Genome-wide identification and characterization of the MAPKKK gene family in A.pumila(1)A total of 143 MAPKKK genes were identified from A.pumila genome,which were evolutionarily divided into three subfamilies:RAF(87),MEKK(36)and ZIK(20).(2)The gene structures of most genes in the RAF and ZIK subfamilies are conserved,while the MEKK subfamily has undergone substantial exon acquisition and deletion during evolution;RAF has 10 motifs,MEKK contains 9,and ZIK contains only 8.(3)There were 74 and 72 collinear genes between A.thaliana,A.lyrata and A.pumila,respectively,indicating that this family expanded obviously in A.pumila genome.(4)Evolutionary analysis showed that there were 64 pairs of duplicated genes,and dominated by purifying selection(both Ka/Ks less than 1).(5)ApMAPKKK genes displayed tissue-specific expression profiles and four expression characteristics under high salt stress(250 m M Na Cl),with significant upregulation of ApMEKK17-1/2 and ApMEKK18-1/2.2.Expression characteristics of ApMEKK18-2 gene(1)ApMEKK18-2 expression was rapidly induced under 250 m M Na Cl,20%PEG6000 and 300 m M mannitol stresses in a short period of time,but ApMEKK18-2 showed a late response pattern in response to1μM ABA treatment.(2)ApMEKK18-2 was highly expressed in tissues such as flowers,fruit stalks,flower buds and young roots,but lowly expressed in tissues such as embryo,rosette leaves,cauline leaves,cotyledons,fruit pods and seeds.(3)ApMEKK18-2 was highly expressed at the early pod setting and maturity stages of growth and development,with lowly expression at the shoot,flower initiation and mid-pod setting stages.3.Transgenic research of ApMEKK18-2 gene(1)The full-length CDS of ApMEKK18-2 gene is 1020 bp,which encodes a peptide of 339 amino acids.(2)Overexpression of ApMEKK18-2 in A.thaliana alleviated seed germination under abiotic stresses.(3)Transgenic studies in rice revealed that oveexpresion of ApMEKK18-2 in rice alleviated the inhibition of rice germination and seedling growth under ABA stress,enhanced rice seed germination under salt stress,and improved the survival rate of rice stressed under 150 m M.In summary,we identified 143 MAPKKK genes from A.pumila and cloned the ApMEKK18-2 gene.Genetic engineering studies showed that overexpression of ApMEKK18-2 in A.thaliana and rice can enhance plant tolerance to salt stress.